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At least 667 records · Page 37Linked to original sources
PubMed1985

Transfer of tubulointerstitial nephritis in the Brown Norway rat with anti-tubular basement membrane antibody: quantitation and kinetics of binding and effect of decomplementation.

Lewis (LEW) rats immunized with Brown Norway (BN) rat renal basement membrane (RBM) and adjuvants produce high titer circulating anti-BN tubular basement membrane (TBM) antibodies, in addition to developing an autoimmune cell-mediated form of nodular tubulointerstitial nephritis (TIN). This immune LEW serum, which reacted with BN TBM but not LEW TBM by immunofluorescence, was capable of passively transferring TIN as early as 24 hr after administration of volumes as low as 3 ml i.v. to normal BN recipients, producing focal lesions histologically and immunopathologically similar but less extensive than those studied previously in this strain after active immunization with heterologous RBM. In contrast, a total of 45 ml of serum (in multiple doses) from BN rats immunized with bovine RBM and adjuvants produced only one small lesion of TIN in a recipient BN rat. This difference in serum transferability of anti-TBM-associated TIN appears to relate to quantitative differences in anti-particulate and soluble (collagenase-extracted) BN RBM antigen reactivity measured by radioimmunoassay. Paired-label quantitative studies of passively transferred LEW anti-BN RBM IgG demonstrated a slow accumulation of renal-bound antibody over 6 days, and corresponded with kidney elution and immunofluorescence studies after transfer of immune LEW sera to normal BN rats. Approximately 167 micrograms of kidney-fixing antibody per gram of kidney were calculated to be required for the development of the earliest cellular infiltration. C3 depletion with cobra venom factor greatly diminished the development of destructive TIN lesions associated with multinucleate giant cells after passive transfer of LEW anti-BN RBM antibody to BN rats. This study, using immune LEW sera containing high levels of anti-BN RBM antibody, has defined and quantitated a role for anti-TBM antibody and complement in the initiation of TIN in BN rats.

Animals↗
PubMed1996

Modeling alternatives for cerebral carbon-11-iomazenil kinetics.

UNLABELLED: The in vivo binding kinetics of [11C]iomazenil, a central benzodiazepine antagonist, were analyzed using PET and compartmental modeling. This method is of interest because it allows validation of the SPECT tracer [123I]iomazenil. METHODS: The experimental protocol consisted of serial PET imaging following a single bolus injection of the serial PET imaging following a single bolus injection of the radioligand. Imaging was performed on five healthy young volunteers over 106 min. The tissue time-activity curves of various brain regions were analyzed with models consisting of two (K1, k2") and three (K1, k2', k3', k4) compartments. Some of the methods use simultaneous fitting of the data from multiple brain regions coupled with common parameters. Distribution volumes and k3-based parameters [(K1/k2') k3' and k3')] were chosen to represent receptor density. Goodness of fit was assessed with F-test statistics and chi-square analysis. RESULTS: Compared with the two-compartment model, goodness of fit was significantly improved by all three-compartment configurations. Of the three-compartment models, goodness of fit was similar for the configurations with K1/k2', k4 or no parameter coupled, and slightly worse when both parameters were coupled. The most reliable estimates of receptor density were obtained from the specific distribution volumes (DVs) calculated with the three-compartment model, and the coupling of k4 or both k4 and K1/k2'. Due to oversimplification of the kinetics, the DV values calculated with the two-compartment model were underestimated. CONCLUSION: Reliable quantitative information regarding benzodiazepine receptor density following bolus injection of iomazenil is best obtained by tracer kinetic modeling that uses a three-compartment model and parameter coupling.

Adult↗
PubMed2005

Structural dynamics of the DnaK-peptide complex.

The molecular chaperone DnaK recognizes and binds substrate proteins via a stretch of seven amino acid residues that is usually only exposed in unfolded proteins. The binding kinetics are regulated by the nucleotide state of DnaK, which alternates between DnaK.ATP (fast exchange) and DnaK.ADP (slow exchange). These two forms cycle with a rate mainly determined by the ATPase activity of DnaK and nucleotide exchange. The different substrate binding properties of DnaK are mainly attributed to changes of the position and mobility of a helical region in the C-terminal peptide-binding domain, the so-called LID. It closes the peptide-binding pocket and thus makes peptide binding less dynamic in the ADP-bound state, but does not (strongly) interact with peptides directly. Here, we address the question if nucleotide-dependent structural changes may be observed in the peptide-binding region that could also be connected to peptide binding kinetics and more importantly could induce structural changes in peptide stretches using the energy available from ATP hydrolysis. Model peptides containing two cysteine residues at varying positions were derived from the structurally well-documented peptide NRLLLTG and labelled with electron spin sensitive probes. Measurements of distances and mobilities of these spin labels by electron paramagnetic resonance spectroscopy (EPR) of free peptides or peptides bound to the ATP and ADP-state of DnaK, respectively, showed no significant changes of mobility nor distance of the two labels. This indicates that no structural changes that could be sensed by the probes at the position of central leucine residues located in the center of the binding region occur due to different nucleotide states. We conclude from these studies that the ATPase activity of DnaK is not connected to structural changes of the peptide-binding pocket but rather only has an effect on the LID domain or other further remote residues.

Adenosine Triphosphate↗
PubMed1983

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase.

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.

Chloride Peroxidase↗
PubMed2003

The kinetics of binding to p38MAP kinase by analogues of BIRB 796.

BIRB 796, a member of the N-pyrazole-N'-naphthly urea class of p38MAPK inhibitors, binds to the kinase with both slow association and dissociation rates. Prior to binding, the kinase undergoes a reorganization of the activation loop exposing a critical binding domain. We demonstrate that, independent of the loop movement, association rates are governed by low energy conformations of the inhibitor and polar functionality on the tolyl ring. As anticipated, the dissociation rates of the inhibitors from the kinase are slowed by lipophilic and hydrogen bond interactions. The value of structure-kinetic relationships (SKR) in drug design is discussed.

Autoimmune Diseases↗
PubMed2005

Association of serotonin 5-HT2A receptor binding and the T102C polymorphism in depressed and healthy Caucasian subjects.

Serotonin 5-HT2A receptor (5-HT2A) binding is reported to be altered in individuals with suicidal behavior, mood disorders, and aggressive-impulsive traits. Genetic association with major depression, suicidal behavior, and aggressive-impulsive traits has not been established. This study examines the possible association of the 5-HT2A gene C102T polymorphism with the receptor binding kinetics, and clinical overt phenotypes. The study population included 63 healthy volunteers and 152 subjects with mood disorders, 56 of whom had a history of suicide attempts. All were Caucasian. Platelet 5-HT2A binding kinetics (Bmax and KD) were assayed and adjusted for seasonal variation. All subjects were genotyped for the T102C polymorphism. Clinical phenotype was determined by structured clinical interview. The TT genotype was associated with higher Bmax in all subjects (F=3.53, df=2,211; p=0.03), controlling for diagnosis. Bonferroni-adjusted post hoc testing showed higher binding in the TT compared with TC genotype in the control group (F=7.56, df=2,60, p=0.001), but not in the mood-disordered subjects. No difference was found in genotype and allele distribution between the mood-disordered subjects, with and without suicide attempt history, and controls. Bmax was not related to a diagnosis of mood disorders. The TT genotype appears associated with higher platelet 5-HT2A Bmax in the healthy population, but this genotypic effect appears absent in mood disorders and unrelated to psychopathology.

Adult↗
PubMed2005

Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization.

Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.

Carcinoma, Hepatocellular↗
PubMed1999

Surface plasmon resonance biosensor studies of human wild-type and mutant lecithin cholesterol acyltransferase interactions with lipoproteins.

Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.

Apolipoprotein A-I↗
PubMed2005

Peripheral-type benzodiazepine receptor binding sites in platelets of patients with panic disorder associated to separation anxiety symptoms.

RATIONALE: Although it is still a matter of debate whether panic disorder (PD) and separation anxiety (SA) are associated or causally linked disorders, some investigators have suggested that SA may be a specific subtype of panic-agoraphobic spectrum. Several psychiatric disorders, including PD, are associated with lower levels of peripheral-type benzodiazepine receptor (PBR). OBJECTIVES: The aim of the present study was to evaluate the kinetic binding parameters of the specific PBR ligand, PK 11195, in platelets from patients with PD in relation to the presence and severity of adulthood SA. METHODS: Using the specific radioligand, [(3)H] PK 11195, the kinetic binding parameters of PBR were determined on platelet membranes of 27 adult outpatients with a DSM-IV diagnosis of PD and 18 healthy controls. Patients were assessed with the SCID-I, the Panic Disorder Severity Scale, the Structured Clinical Interview for Separation Anxiety Symptoms and the Adult Separation Anxiety Checklist. RESULTS: PD patients had significantly lower PBR density than controls. However, the lower density was only evident in the subgroup of PD patients who also fulfilled the DSM-IV criteria for adult separation anxiety disorder. PBR density was negatively correlated with each of the two SA scales total scores. CONCLUSIONS: Patients with SA symptoms had significantly lower densities of PBRs. PBR expression might become a useful biological marker of these two associated conditions.

Adult↗
PubMed2004

Challenges and artifacts in quantitative photobleaching experiments.

Confocal fluorescence recovery after photobleaching (FRAP) is today the prevalent tool when studying the diffusional and kinetic properties of proteins in living cells. Obtaining quantitative data for diffusion coefficients via FRAP, however, is challenged by the fact that both bleaching and scanning take a finite time. Starting from an experimental case, it is shown by means of computer simulations that this intrinsic temporal limitation can lead to a gross underestimation of diffusion coefficients. Determining the binding kinetics of proteins to membranes with FRAP is further shown to be severely hampered by additional diffusional contributions, e.g. diffusion-limited binding. In some cases, the binding kinetics may even be masked entirely by diffusion. As current efforts to approach biological problems with biophysical models have to rely on experimentally determined model parameters, e.g. binding rates and diffusion constants, it is proposed that the accuracy in evaluating FRAP measurements can be improved by means of accompanying computer simulations.

Fluorescence Recovery After Photobleaching↗
PubMed2006

Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor.

Listeria adhesion protein (LAP) is an important adhesion factor in Listeria monocytogenes and interacts with its cognate receptor, mammalian heat shock protein 60 (Hsp60). The genetic identity of LAP was determined to be alcohol acetaldehyde dehydrogenase (Aad). A recombinant Escherichia coli strain expressing aad confirmed the involvement of Aad in adhesion to Caco-2 cells. Binding kinetics (ka) of recombinant LAP (rLAP) to Hsp60 was examined in a surface plasmon resonance sensor and was determined to be 5.35 x 10(8) M(-1) s(-1) and it was equivalent to the binding of anti-Hsp60 antibody (ka = 2.15 x 10(9) M(-1) s(-1)) to Hsp60. In contrast, Internalin B, an adhesion/invasion protein from L. monocytogenes, used as a control, had binding kinetics (ka) of only 2.9 x 10(6) M(-1) s(-1). The KD value of rLAP was 1.68 x 10(-8) M, which was significantly lower than Internalin B (KD = 6.5 x 10(-4) M). These results suggest that Hsp60 has significantly higher avidity for anti-Hsp60 antibody and LAP than Internalin B. In summary, LAP is identified as an alcohol acetaldehyde dehydrogenase and binding of recombinant E. coli to Caco-2 cells or rLAP to Hsp60 protein was found to be highly specific.

Adhesins, Bacterial↗
PubMed2005

Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5.

The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor). In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously. Kinetic investigations of the redox couple FdRmor/Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fdmor in this reaction, compared with the NADH-dependent reduction of cytochrome c. The quite low Km (5.3 +/- 0.3 nm) of FdRmor for Fdmor, measured with NBT as the electron acceptor, indicated high specificity. The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins. Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form, which has previously not been possible for wild-type P450mor. The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under any conditions. Pyridine, metyrapone and different azole compounds generated type II binding spectra and the Kd values determined for these substances suggested that P450mor might have a preference for more bulky and/or hydrophobic molecules. The purified recombinant proteins FdRmor, Fdmor and P450mor were used to reconstitute the homologous P450-containing mono-oxygenase, which was shown to convert morpholine.

Catalysis↗
PubMed1995

PET imaging studies of dopamine D2 receptors: comparison of [18F]N-methylspiperone and the benzamide analogues [18F]MABN and [18F]MBP in baboon brain.

A series of positron emission tomography (PET) imaging studies was conducted in a baboon with the benzamide derivatives [18F]2,3-dimethoxy-N-[9-(4-fluorobenzyl)-9-azabicyclo[3.3.1]non an-3 beta-yl]benzamide ([18F]MABN) and [18F]2,3-dimethoxy-N-[1-(4-fluorobenzyl)piperidin-4-yl]be nza mide ([18F]MBP). Studies were also conducted with the butyrophenone [18F]N-methylspiperone (NMSP) for comparison. Tissue-time activity curves of [18F]MABN are similar to those of [18F]NMSP since both compounds displayed approximately the same uptake in the basal ganglia and displayed irreversible binding kinetics in vivo. However, the rapid rate of clearance from the cerebellum and high basal ganglia:cerebellum ratio of [18F]MABN indicate that this compound has a much lower amount of nonspecific binding than [18F]NMSP. [18F]MBP displayed a higher uptake in the basal ganglia relative to [18F]NMSP and [18F]MABN and exhibited reversible binding kinetics in vivo. This property of [18F]MBP is desirable since the uptake of radioactivity in D2-rich ligands is less likely to be influenced by changes in cerebral blood flow. The current data suggest that both [18F]MABN and [18F]MBP are promising ligands for studying dopamine D2 receptors with PET.

Animals↗
PubMedDate not supplied

Analysis of the temporal compressibility of breast tumour marker assays: development of a "near patient" assay.

The aim of this study was to investigate whether immunoassays for circulating MUC1 antigen in breast cancer could be compressed in time so that serum level results would be made available during the time of the patient's visit to clinic. Two assays were used: -The EMCA (Euro DPC) is a liquid phase immunoassay and the ELSA CA15-3 (CIS) is a double determinant solid phase immunoradiometric assay. The effects of shortened incubation times were investigated by assaying standards and unknown samples and comparing the results with those using the standard kit protocols. The binding kinetics of the monoclonal antibodies employed in the assays were analysed separately. We conclude that the EMCA assay can be shortened to 35 min and we have attributed this to the fast binding kinetics inherent in a liquid phase assay. This shortened assay may produce the basis for a useful "near patient" assay. By comparison, the solid phase ELSA CA15-3 assay cannot be compressed without loss in assay performance.

Antibodies, Monoclonal↗
PubMed1998

Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA.

Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially. Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude. Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300. Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.

Animals↗
PubMed2002

Kinetic and binding analysis of the catalytic involvement of ribose moieties of a trans-acting delta ribozyme.

We have identified ribose 2'-hydroxyl groups (2'-OHs) that are critical for the activity of a trans-cleaving delta ribozyme derived from the antigenomic strand of the hepatitis delta virus. Initially, an RNA-DNA mixed ribozyme composed of 26 deoxyribo- (specifically the nucleotides forming the P2 stem and the P4 stem-loop) and 31 ribonucleotides (those forming the catalytic center) was engineered. This mixed ribozyme catalyzed the cleavage of a small substrate with kinetic parameters virtually identical to those of the all-RNA ribozyme. The further substitution of deoxyribose for ribose residues permitted us to investigate the contribution of all 2'-OHs to catalysis. Determination of the kinetic parameters for the cleavage reaction of the resulting ribozymes revealed (i) 10 2'-OH groups appear to be important in supporting the formation of several hydrogen bonds within the catalytic core, (ii) none of the important 2'-OHs seem to coordinate a magnesium cation, and (iii) 1 of the tested RNA-DNA mixed polymers appeared to stabilize the ribozyme-substrate transition-state complex, resulting in an improvement over the all-RNA counterpart. The contribution of the 2'-OHs to the catalytic mechanism is discussed, and differences with the crystal structure of a genomic delta self-cleaved product are explained. Clearly, the 2'-OHs are essential components of the network of interactions involved in the formation of the catalytic center of the delta ribozyme.

Base Sequence↗